Illumination of optical analytical devices

ABSTRACT

Optical analytical devices and their methods of use are provided. The devices are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices include optical waveguides for illumination of the optical reactions. The devices further provide for the efficient coupling of optical excitation energy from the waveguides to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination using features such as spectra, amplitude, and time resolution, or combinations thereof. The devices of the invention are well suited for miniaturization and high throughput.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/805,801, filed on Mar. 1, 2020, now U.S. Pat. No. 11,137,532, whichis a continuation of U.S. patent application Ser. No. 16/030,421, filedon Jul. 9, 2018, now U.S. Pat. No. 10,578,788, which is a continuationof U.S. patent application Ser. No. 14/945,186, filed on Nov. 18, 2015,now U.S. Pat. No. 10,018,764, which is a continuation of U.S. patentapplication Ser. No. 14/107,730, filed on Dec. 16, 2013, now U.S. Pat.No. 9,223,084, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/738,637, filed on Dec. 18, 2012, thedisclosures of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

In analytical systems, the ability to increase the number of analysesbeing carried out at any given time by a given system has been a keycomponent to increasing the utility and extending the lifespan of suchsystems. In particular, by increasing the multiplex factor of analyseswith a given system, one can increase the overall throughput of thesystem, thereby increasing its usefulness while decreasing the costsassociated with that use.

In optical analyses, increasing multiplex often poses increaseddifficulties, as it may require more complex optical systems, increasedillumination or detection capabilities, and new reaction containmentstrategies. In some cases, systems seek to increase multiplex by manyfold, and even orders of magnitude, which further implicate theseconsiderations. Likewise, in certain cases, the analytical environmentfor which the systems are to be used is so highly sensitive thatvariations among different analyses in a given system may not betolerable. These goals are often at odds with a brute force approach ofsimply making systems bigger and of higher power, as such steps oftengive rise to even greater consequences, e.g., in inter reactioncross-talk, decreased signal to noise ratios resulting from either orboth of lower signal and higher noise, and the like. It would thereforebe desirable to provide analytical systems that have substantiallyincreased multiplex for their desired analysis, and particularly for usein highly sensitive reaction analyses, and in many cases, to do so whileminimizing negative impacts of such increased multiplex.

Conventional optical systems employ complex optical trains that direct,focus, filter, split, separate, and detect light to and/or from thesample materials. Such systems typically employ an assortment ofdifferent optical elements to direct, modify, and otherwise manipulatelight directed to and/or received from a reaction site. Such systems aretypically complex and costly and tend to have significant spacerequirements. For example, typical systems employ mirrors and prisms indirecting light from its source to a desired destination. Additionally,such systems may include light-splitting optics such as beam-splittingprisms to generate two or more beams from a single original beam.

There is a continuing need to increase the performance of analyticalsystems and reduce the cost associated with manufacturing and using thesystem. In particular, there is a continuing need to increase thethroughput of analytical systems. There is a continuing need to reducethe size and complexity of analytical system. There is a continuing needfor analytical systems that have flexible configurations and are easilyscalable.

SUMMARY OF THE INVENTION

The instant invention addresses these and other problems by providing inone aspect an analytical device comprising an optical waveguide forillumination of nanometer-scale apertures. The waveguide comprises anoptical core and a cladding. The device further comprises, in someembodiments, a metallic layer disposed on a surface of the cladding, aplurality of nanometer-scale apertures disposed in the metallic layer insufficient proximity to the optical waveguide to be illuminated by anevanescent field emanating from the waveguide when optical energy ispassed through the optical core, and a plurality of local fieldenhancement elements associated with the plurality of apertures.

In some embodiments, the local field enhancement elements comprise ahigh dielectric material or metal in the vicinity of the apertures.

In some embodiments, the high dielectric material or metal of theinstant analytical device is arranged in a geometric pattern around theapertures.

In some embodiments, the geometric pattern is selected from the groupconsisting of a circle, a series of concentric circles, a C aperture, atriangle pair, and a diamond. In specific embodiments, the highdielectric material or metal is Al₂O₃, copper, silver, gold, oraluminum. In more specific embodiments, the high dielectric material ormetal is Al₂O₃. In other more specific embodiments, the high dielectricmaterial or metal is copper.

In some embodiments, the apertures of the device are recessed into thecladding. In some embodiments, the thickness of the cladding between theoptical core and the metallic layer decreases in the vicinity of theapertures. In specific embodiments, the thickness of the cladding isfrom 150 to 300 nm. In more specific embodiments, the thickness of thecladding is about 200 nm.

In certain embodiments, the local field enhancement elements comprisethe shape of the nanometer-scale apertures. In specific embodiments, theshape of the nanometer-scale apertures is selected from the groupconsisting of a C aperture, a triangle pair, and a diamond.

In another aspect, the invention provides an analytical devicecomprising an optical waveguide comprising an optical core and acladding, a metallic layer disposed on the surface of the cladding; anda plurality of nanometer-scale apertures disposed in the metallic layerin sufficient proximity to the optical waveguide to be illuminated by anevanescent field emanating from the waveguide when optical energy ispassed through the optical core, wherein the optical core has athickness, a width, and a cross-sectional area, and wherein thecross-sectional area is decreased at locations where the evanescentfield illuminates the apertures.

In some embodiments, the cross-sectional area of the optical core isdecreased by adiabatic tapers.

In some embodiments, the thickness of the optical core is maintained,and the cross-sectional area is decreased by decreasing the width of theoptical core. In preferred embodiments, the optical energy is transverseelectric polarized light.

In some embodiments, the width of the optical core is maintained, andthe cross-sectional area is decreased by decreasing the thickness of theoptical core. In preferred embodiments, the optical energy is transversemagnetic polarized light.

In some embodiments, the analytical device further comprises a pluralityof local field enhancement elements associated with the plurality ofapertures, as described above.

In another aspect, the invention provides an analytical devicecomprising an optical waveguide comprising a plurality of optical coresand a cladding, a plurality of nanometer-scale apertures disposed on asurface of the device in sufficient proximity to the optical waveguideto be illuminated by an evanescent field emanating from the waveguidewhen optical energy is passed through the plurality of optical cores,and a plurality of detectors optically coupled to the plurality ofnanometer-scale apertures, wherein the optical cores are not in directalignment between the nanometer-scale apertures and their opticallycoupled detectors.

In preferred embodiments, the plurality of nanometer-scale apertures areilluminated by an evanescent field emanating from at least two opticalcores. In other preferred embodiments, the analytical device furthercomprises an opaque layer disposed between the optical waveguide and theplurality of optical detectors, wherein the opaque layer comprises aplurality of openings in direct alignment with the nanometer-scaleapertures and their optically coupled detectors. In still otherpreferred embodiments, the device further comprises a plurality of localfield enhancement elements associated with the plurality of apertures,as described above.

In yet another aspect, the invention provides an analytical devicecomprising an optical waveguide comprising an optical core and acladding, and a plurality of nanometer-scale apertures disposed on asurface of the device in sufficient proximity to the optical waveguideto be illuminated by an evanescent field emanating from the waveguidewhen optical energy of a defined wavelength is passed through theoptical core, wherein the wavelength of the optical energy is modulatedas it passes through the optical core.

In some embodiments of the analytical device, the optical waveguidecomprises a non-linear optical material. In preferred embodiments, thenon-linear optical material is placed periodically within the opticalcore. In other preferred embodiments, the non-linear optical material isplaced within the cladding.

In particularly preferred embodiments, the wavelength conversion iseffected through phase matching. In other particularly preferredembodiments, the wavelength conversion is effected throughelectro-optical effects.

In some embodiments, the optical energy is modulated by second harmonicgeneration. In other embodiments, the optical energy is modulated bythird harmonic generation.

In some embodiments, the optical energy is modulated by opticalparametric amplification. In some embodiments, the device furthercomprises a plurality of local field enhancement elements associatedwith the plurality of apertures, as described above.

In some embodiments, the analytical device further comprises a pluralityof analytes disposed in analyte regions within the plurality ofnanometer-scale apertures. In specific embodiments, the plurality ofanalytes comprise a plurality of biological samples. In preferredembodiments, the plurality of biological samples comprise a plurality ofnucleic acids. In more preferred embodiments, the analytical devicecomprises at least 1,000, at least 10,000, at least 100,000, at least1,000,000, or at least 10,000,000 nanometer-scale apertures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B schematically illustrate an exemplary nucleic acidsequencing process that can be carried out using aspects of theinvention.

FIG. 2 provides a schematic block diagram of an integrated analyticaldevice.

FIG. 3A provides a schematic of excitation spectra for two signal eventsand an indicated narrow band excitation illumination, while FIG. 3Bschematically illustrates the resulting detected signal based upon thenarrow band illumination of the two signal events.

FIG. 4 schematically illustrates the signal profiles for each of fourfluorescent labeling groups, overlain with each of two different filterprofiles.

FIG. 5 schematically illustrates an integrated analytical device fordetecting signals from a 4-color sequencing reaction.

FIG. 6 schematically illustrates signal traces for a two-color,two-amplitude sequence-by-synthesis reaction.

FIG. 7A illustrates an analytical device containing a waveguide core,cladding, metallic/opaque layer, and a material of high dielectric ormetal in the vicinity of the nanometer-scale aperture, as viewed in thex-z plane. FIG. 7B illustrates an alternative device structure, having ageometric pattern of high dielectric material or metal surrounding theaperture. FIG. 7C illustrates another alternative device structure,having a decreased cladding thickness in the regions of the waveguideadjacent to the aperture and/or an increased cladding thickness in theregions not adjacent to the aperture. The device of FIG. 7C additionallyincludes a high dielectric material or metal in the vicinity of theaperture.

FIGS. 8A and 8B illustrate an analytical device where the thickness ofthe cladding is decreased in the regions of waveguide adjacent to theaperture, and the device additionally contains a stray light terminationmaterial. FIG. 8A shows a top view in the x-y plane with the opaque,metallic layer omitted from the view. FIG. 8B shows a cross-sectionalview in the y-z plane.

FIGS. 9A-9C provide a schematic of the design used in simulations ofcoupling efficiencies. FIG. 9A shows a top view of the simulated devicein the x-y plane with the opaque, metallic layer omitted from the view.FIG. 9B shows a side view of the simulated device in the x-z plane. FIG.9C shows a cross-sectional view of the simulated device in the y-zplane.

FIGS. 10A and 10B illustrate an example of the dimensional modulation ofthe waveguides.

FIGS. 11A and 11B illustrate an example of a device with a “slotted”waveguide. FIG. 11A: cross-sectional view; FIG. 11B: top-down view.

FIGS. 12A and 12B show the configuration and optical modes of awaveguide containing a non-linear optical material in the core (FIG.12A) and in part of the cladding (FIG. 12B).

FIGS. 13A and 13B illustrate configurations of waveguides containingnon-linear optical materials.

FIGS. 14A and 14B schematically illustrate a waveguide containing awavelength conversion element just after the coupler (FIG. 14A) andanother exemplary waveguide with independently addressable SHG elements(FIG. 14B).

DETAILED DESCRIPTION OF THE INVENTION

Integrated Optical Detection Devices

Multiplexed optical analytical systems are used in a wide variety ofdifferent applications. Such applications can include the analysis ofsingle molecules, and can involve observing, for example, singlebiomolecules in real time as they carry out reactions. For ease ofdiscussion, such multiplexed systems are discussed herein in terms of apreferred application: the analysis of nucleic acid sequenceinformation, and particularly, single molecule nucleic acid sequenceanalysis. Although described in terms of a particular application, itshould be appreciated that the applications for the devices and systemsdescribed herein are of broader application.

In the context of single molecule nucleic acid sequencing analyses, asingle immobilized nucleic acid synthesis complex, comprising apolymerase enzyme, a template nucleic acid, whose sequence is beingelucidated, and a primer sequence that is complementary to a portion ofthe template sequence, is observed in order to identify individualnucleotides as they are incorporated into the extended primer sequence.Incorporation is typically monitored by observing an opticallydetectable label on the nucleotide, prior to, during, or following itsincorporation. In some cases, such single molecule analyses employ a“one base at a time approach”, whereby a single type of labelednucleotide is introduced to and contacted with the complex at a time.Upon reaction, unincorporated nucleotides are washed away from thecomplex, and the labeled incorporated nucleotides are detected as a partof the immobilized complex.

In some instances, only a single type of nucleotide is added to detectincorporation. These methods then require a cycling through of thevarious different types of nucleotides (e.g., A, T, G and C) to be ableto determine the sequence of the template. Because only a single type ofnucleotide is contacted with the complex at any given time, anyincorporation event is, by definition, an incorporation of the contactednucleotide. These methods, while somewhat effective, generally sufferfrom difficulties when the template sequence includes multiple repeatednucleotides, as multiple bases may be incorporated that areindistinguishable from a single incorporation event. In some cases,proposed solutions to this issue include adjusting the concentrations ofnucleotides present to ensure that single incorporation events arekinetically favored.

In other cases, multiple types of nucleotides are added simultaneously,but the nucleotides are distinguishable by the presence on each type ofnucleotide a different optical label. Accordingly, such methods can usea single step to identify a given base in the sequence. In particular,all four nucleotides, each bearing a distinguishable label, are added tothe immobilized complex. The complex is then interrogated to identifywhich type of base was incorporated, and as such, the next base in thetemplate sequence.

In some cases, these methods only monitor the addition of one base at atime, and as such, they (and in some cases, the single nucleotidecontact methods) require additional controls to avoid multiple basesbeing added in any given step, and thus being missed by the detectionsystem. Typically, such methods employ terminator groups on thenucleotide that prevent further extension of the primer once onenucleotide has been incorporated. These terminator groups are typicallyremovable, allowing the controlled re-extension after a detectedincorporation event. Likewise, in order to avoid confounding labels frompreviously incorporated nucleotides, the labeling groups on thesenucleotides are typically configured to be removable or otherwiseinactivatable.

In another example, single molecule primer extension reactions aremonitored in real time, to identify the continued incorporation ofnucleotides in the extension product to elucidate the underlyingtemplate sequence. In such single molecule real time (or SMRT™)sequencing, the process of incorporation of nucleotides in apolymerase-mediated template-dependent primer extension reaction ismonitored as it occurs. In preferred aspects, the template/polymeraseprimer complex is provided, typically immobilized, within anoptically-confined region, such as a zero mode waveguide (ZMW), orproximal to the surface of a transparent substrate, optical waveguide,or the like (see e.g., U.S. Pat. Nos. 6,917,726, 7,170,050, and7,935,310, the full disclosures of which are hereby incorporated byreference herein in their entirety for all purposes). Theoptically-confined region is illuminated with an appropriate excitationradiation for the fluorescently-labeled nucleotides that are to be used.Because the complex is within an optically confined region, or verysmall illumination volume, only the reaction volume immediatelysurrounding the complex is subjected to the excitation radiation.Accordingly, those fluorescently labeled nucleotides that areinteracting with the complex, e.g., during an incorporation event, arepresent within the illumination volume for a sufficient time to identifythem as having been incorporated.

A schematic illustration of this sequencing process is shown in FIGS. 1Aand 1B. As shown in FIG. 1A, an immobilized complex 102 of a polymeraseenzyme, a template nucleic acid and a primer sequence are providedwithin an observation volume (as shown by dashed line 104) of an opticalconfinement, of e.g., a zero mode waveguide 106. As an appropriatenucleotide analog, e.g., nucleotide 108, is incorporated into thenascent nucleic acid strand, it is illuminated for an extended period oftime corresponding to the retention time of the labeled nucleotideanalog within the observation volume during incorporation. Theincorporation reaction thus produces a signal associated with thatretention, e.g., signal pulse 112 as shown by the A trace in FIG. 1B.Once incorporated, the label that was attached to the polyphosphatecomponent of the labeled nucleotide analog, is released. When the nextappropriate nucleotide analog, e.g., nucleotide 110, is contacted withthe complex, it too is incorporated, giving rise to a correspondingsignal 114 in the T trace of FIG. 1B. By monitoring the incorporation ofbases into the nascent strand, as dictated by the underlyingcomplementarity of the template sequence, long stretches of sequenceinformation of the template can be obtained.

The above sequencing reaction may be incorporated into a device,typically an integrated analytical device, that provides for thesimultaneous observation of multiple sequencing reactions, ideally inreal time. While the components of each device, and the configuration ofthe devices in the system, may vary, each integrated analytical devicetypically comprises, at least in part, the general structure shown as ablock diagram in FIG. 2 . As shown, an integrated analytical device 200typically includes a reaction cell 202, in which the reactants aredisposed and from which the optical signals emanate. The analysis systemfurther includes a detector element 220, which is disposed in opticalcommunication with the reaction cell 202. Optical communication betweenthe reaction cell 202 and the detector element 220 may be provided by anoptical train 204 comprised of one or more optical elements generallydesignated 206, 208, 210 and 212 for efficiently directing the signalfrom the reaction cell 202 to the detector 220. These optical elementsmay generally comprise any number of elements, such as lenses, filters,gratings, mirrors, prisms, refractive material, or the like, or variouscombinations of these, depending upon the specifics of the application.By integrating these elements into a single device architecture, theefficiency of the optical coupling between the reaction cell and thedetector is improved. Examples of integrated analytical systems,including various approaches for illuminating the reaction cell anddetecting optical signals emitted from the reaction cell, are describedin U.S. Patent Application Publication Nos. 2012/0014837, 2012/0019828,and 2012/0021525, which are each incorporated by reference herein intheir entireties for all purposes.

Conventional analytical systems typically measure multiple spectrallydistinct signals or signal events and must therefore utilize complexoptical systems to separate and distinctly detect those different signalevents. The optical path of an integrated device may be simplified,however, by a reduction in the amount or number of spectrallydistinguishable signals that are detected. Such a reduction is ideallyeffected, however, without reducing the number of distinct reactionevents that can be detected. For example, in an analytical system thatdistinguishes four different reactions based upon four differentdetectable signal events, where a typical system would assign adifferent signal spectrum to each different reaction, and thereby detectand distinguish each signal event, in an alternative approach, fourdifferent signal events would be represented at fewer than fourdifferent signal spectra, and would, instead, rely, at least in part, onother non-spectral distinctions between the signal events.

For example, a sequencing operation that would conventionally employfour spectrally distinguishable signals, e.g., a “four-color” sequencingsystem, in order to identify and characterize the incorporation of eachof the four different nucleotides, would, in the context of analternative configuration, employ a one-color or two-color analysis,e.g., relying upon a signals having only one or two distinct ordistinguished spectral signals. However, in such an alternativeconfiguration, this reduction in reliance on signal spectral complexitydoes not come at the expense of the ability to distinguish signals frommultiple, i.e., a larger number of different signal producing reactionevents. In particular, instead of relying solely on signal spectrum todistinguish reaction events, such an alternative configuration may relyupon one or more signal characteristics other than emission spectrum,including, for example, signal intensity, excitation spectrum, or bothto distinguish signal events from each other.

In one particular alternative configuration, the optical paths in anintegrated analytical device may thus be simplified by utilizing signalintensity as a distinguishing feature between two or more signal events.In its simplest iteration, and with reference to an exemplary sequencingprocess, two different types of nucleotides would bear fluorescentlabels that each emit fluorescence under the same excitationillumination, i.e., having the same or substantially overlappingspectral band, and thus would provide benefits of being excited using asingle excitation source and beam. The resulting signals from eachfluorescent label would have distinct signal intensities or amplitudesunder that same illumination, and would be distinguishable by theirrespective signal amplitudes. These two signals could have partially orentirely overlapping emission spectra, but separation of the signalsbased upon any difference in emission spectrum would be unnecessary.

Accordingly, for analytical systems using two or more signal events thatdiffer in signal amplitude, the integrated analytical devices of suchsystems can readily benefit through the removal of some or all of thosecomponents that would normally be used to separate spectrally distinctsignals, such as multiple excitation sources and their associatedoptical trains, as well as the color separation optics, e.g., filtersand dichroics, for the signal events, which in many cases, requires atleast partially separate optical trains and detectors for eachspectrally distinct signal. As a result, the optical paths for theseintegrated analytical devices are greatly simplified, allowing placementof detector elements in closer proximity to reaction regions, andimproving overall performance of the detection process for thesedevices.

Provision of signal-producing reactants that will produce differentsignal amplitudes under a particular excitation illumination profile maybe accomplished in a number of ways. For example, different fluorescentlabels may be used that present excitation spectral profiles thatoverlap but include different maxima. As such, excitation at a narrowwavelength will typically give rise to differing signal intensities foreach fluorescent group. This is illustrated in FIG. 3A, which shows theexcitation spectra of two different fluorescent label groups (solid anddashed lines 302 and 304, respectively). When subjected to excitationillumination at the wavelength range shown by vertical lines 306, eachfluorescent label will emit a signal at the corresponding amplitude. Theresulting signal intensities at a given excitation wavelength are thenshown in the bar chart of FIG. 3B, shown as the solid lined and dashedlined bars, respectively. The difference in intensity of these twosignal-producing labels at the given excitation wavelength can then bereadily used to distinguish the two signal events. As will beappreciated, such spectrally indistinct signals would not be easilydistinguishable when occurring simultaneously, as they would result inan additive overlapping signal, unless, as discussed below, suchspectrally indistinct signals result from spectrally distinct excitationwavelengths. As will be appreciated, this same approach may be used withmore than two label groups where the resulting emission at a givenexcitation spectrum have distinguishable intensities or amplitudes.

Similarly, two different fluorescent labeling groups may have the sameor substantially similar excitation spectra, but provide different anddistinguishable signal emission intensities due to the quantum yield orextinction coefficient of those labeling groups.

Further, although described in terms of two distinct fluorescent dyes,it will be appreciated that each different labeling group may eachinclude multiple labeling molecules. For example, each reactant mayinclude an energy transfer dye pair that yields emissions of differingintensities upon excitation with a single illumination source. Forexample, a labeling group may include a donor fluorophore that isexcited at a given excitation wavelength, and an acceptor fluorophorethat is excited at the emission wavelength of the donor, resulting inenergy transfer to the acceptor. By using different acceptors, whoseexcitation spectra overlap the emission spectrum of the donor todiffering degrees, such an approach can produce overall labeling groupsthat emit at different signal amplitudes for a given excitationwavelength and level. Likewise, adjusting the energy transfer efficiencybetween the donor and acceptor will likewise result in differing signalintensities at a given excitation illumination. Examples of theseapproaches are described in U.S. Patent Application Publication No.2010/0255488, the full disclosure of which is incorporated by referenceherein in its entirety for all purposes.

Alternatively, different signal amplitudes may be provided by differentmultiples of signal producing label groups on a given reactant, e.g.,putting a single label molecule on one reactant while putting 2, 3, 4 ormore individual label molecules on a different reactant. The resultingemitted signal will be reflective of the number of labels present on areactant and thus will be indicative of the identity of that reactant.Methods and compositions for coupling multiple labeling groups onreactants, such as nucleotide analogs, are described in, for example,U.S. patent application Ser. No. 13/218,312, filed Aug. 25, 2011, andincorporated by reference herein in its entirety for all purposes.

As described above, integrated analytical devices making use of suchapproaches see a reduction in complexity by elimination of spectraldiscrimination requirements, e.g., using signal amplitude or othernon-spectral characteristics as a basis for signal discrimination.Integrated analytical devices that combine such non-spectraldiscrimination approaches with the more common spectral discriminationapproaches may also provide advantages over more complex spectraldiscrimination systems. By shifting from a “four-color” discriminationsystem to a system that distinguishes signals based upon signalintensity and color, one can still reduce the complexity of the overalloptical system relative to a conventional four-color separation scheme.For example, in an analytical operation that detects four discretereaction events, e.g., in a nucleic acid sequencing analysis, two signalevents may be provided within a given emission/detection spectrum, i.e.,emitting signals within the same spectral window, and the other twoevents within a distinct emission/detection spectrum. Within eachspectral window, the pair of signal events produce distinguishablesignal intensities relative to each other.

For ease of discussion, this concept is described in terms of two groupsof fluorescent signal events, where members of each group differ byfluorescent intensity, and the groups differ by virtue of their emissionspectrum. As will be appreciated, the use of simplified optics systems,e.g., using two detection channels for two distinct emission spectra,does not require that the emission profiles of the two groups of signalsdo not overlap or that the emission spectra of members of each groupperfectly overlap. Instead, in many preferred aspects, more complexsignal profiles may be used where each different signal event possessesa unique emission spectrum, but in a way that each signal will present asignal profile within the two detection channels that is unique, basedupon the signal intensity in each channel.

FIG. 4 schematically illustrates the signal profiles for each of fourfluorescent labeling groups, overlain with each of two different filterprofiles. As shown, four label groups yield emission spectra 402, 404,406 and 408, respectively. While the signals from these four groupspartially overlap each other, they each have different maxima. Whensubjected to a two channel filter scheme, as shown by pass filter lines410 and 412, the signal from each label will produce a unique signalprofile between the two detection channels. In particular, signals arerouted through an optical train that includes two paths that arefiltered according to the spectral profile shown. For each signal,different levels of emitted light will pass through each path and bedetected upon an associated detector. The amount of signal that passesthrough each filter path is dictated by the spectral characteristics ofthe signal.

In the case of the above described mixed-mode schemes, detection systemsmay be provided that include at least two distinct detection channels,where each detection channel passes light within a spectrum that isdifferent from each other channel. Such systems also include a reactionmixture within optical communication of the detection channels, wherethe reaction mixture produces at least three different optical signalsthat each produces a unique signal pattern within the two detectionchannels, as compared to the other optical signals.

In each case, each signal-producing reactant is selected to provide asignal that is entirely distinct from each other signal in at least oneof signal intensity and signal channel. As noted above, signal intensityin a given channel is dictated, in part, by the nature of the opticalsignal, e.g., its emission spectrum, as well as the filters throughwhich that signal is passed, e.g., the portion of that spectrum that isallowed to reach the detector in a given channel. However, signalintensity can also be modulated by random variables, such as orientationof a label group when it is emitting signal, or other variables of theparticular reaction. Accordingly, for a signal's intensity to be assuredof being entirely different from the intensity of another signal withina given channel, in preferred aspects, this variation is accounted for.

With a reduced number of spectrally distinct signal events, thecomplexity of the optical paths for the integrated devices is alsoreduced. FIG. 5 illustrates a not-to-scale example device architecturefor performing optical analyses, e.g., nucleic acid sequencingprocesses, that rely in part on non-spectral discrimination of differingsignals, and optionally, in part on spectral distinction. As shown, anintegrated analytical device 500 includes a reaction region 502 that isdefined upon a first substrate layer 504. As shown, the reaction regioncomprises a well disposed in the substrate surface. Such wells mayconstitute depressions in a substrate surface or apertures disposedthrough additional substrate layers to an underlying transparentsubstrate, e.g., as used in zero mode waveguide arrays (See, e.g., U.S.Pat. Nos. 7,181,122 and 7,907,800).

In the device of FIG. 5 , excitation illumination is delivered to thereaction region from an excitation light source (not shown) that may beseparate from or also integrated into the substrate. As shown, anoptical waveguide (or waveguide layer) 506 may be used to conveyexcitation light (shown by arrows) to the reaction region/well 502,where the evanescent field emanating from the waveguide 506 illuminatesreactants within the reaction region 502. Use of optical waveguides toilluminate reaction regions is described in e.g., U.S. Pat. No.7,820,983 and U.S. Patent Application Publication No. 2012/0085894,which are each incorporated by reference herein in their entireties forall purposes.

The integrated device 500 optionally includes light channelingcomponents 508 to efficiently direct emitted light from the reactionregions to a detector layer 512 disposed beneath the reaction region.The detector layer typically comprises one, or preferably multiple,detector elements 512 a-d, e.g., pixels in an array detector, that areoptically coupled to a given reaction region. Although illustrated as alinear arrangement of pixels 512 a-d, it will be appreciated that suchpixels may be arranged in a grid, n×n square, annular array, or anyother convenient orientation.

Emitted signals from the reaction region 502 that impinge on thesepixels are then detected and recorded. As noted above, an optionalsingle filter layer 510 is disposed between the detector layer and thereaction region, to permit different spectrally distinct signals totravel to different associated pixels 512 a and 512 b in the detectorlayer 512. For example, the portion 510 a of filter layer 510 allowssignals having a first emission spectrum to reach its associated pixels512 a and 512 b, while filter portion 510 b of filter layer 510 allowsonly signals having a distinct second spectrum to reach its associatedpixels 512 c and 512 d.

In the context of a sequencing system exploiting such a configuration,incorporation of two of the four nucleotides would produce signals thatwould be passed through filter portion 510 a to pixels 512 a and 512 b,and blocked by filter portion 510 b. As between these two signals, onesignal would have a signal intensity higher than the other such that thepixels 512 a and 512 b in detector layer 512 would be able to producesignal responses indicative of such differing signal intensities.Likewise, incorporation of the other two nucleotides would producesignals that would be passed through filter portion 510 b to itsassociated pixels 512 c and 512 d, while filter portion 510 a wouldblock those signals from reaching pixels 510 a and 510 b. Again, thesignals associated with these two latter signal events would differbased upon their signal intensities or amplitudes.

The detector layer is then operably coupled to an appropriate circuitry,typically integrated into the substrate, for providing a signal responseto a processor that is optionally included integrated within the samedevice structure or is separate from but electronically coupled to thedetector layer and associated circuitry. Examples of types of circuitryare described in U.S. Patent Application Publication No. 2012/0019828.

As will be appreciated from the foregoing disclosure and FIG. 5 , theintegrated analytical devices described herein do not require the morecomplicated optical paths that are necessary in systems utilizingconventional four-color optics, obviating the need for excessive signalseparation optics, dichroics, prisms, or filter layers. In particular,although shown with a single filter layer, as noted, in optionalaspects, the filter layer could be eliminated or could be replaced witha filter layer that blocks stray light from the excitation source ratherthan distinguishing different emission signals from the reaction region.Even including the filter layer 510, results in simplified and/or moreefficient optics as compared to conventional four-color systems, whichwould require either multilayer filters, or narrow band pass filters,which typically require hybrid layers or composite approaches over eachsubset of pixels, thus blocking signal from reaching three of the fourpixel subsets at any given emission wavelength, resulting in detectionof far fewer photons from each signal event. The optics configurationshown in FIG. 5 , on the other hand, only blocks a smaller portion ofthe overall signal light from reaching the detector. Alternatively, suchconventional systems would require separation and differential directionof all four different signal types, resulting in inclusion of additionaloptical elements, e.g., prisms or gratings, to achieve spectralseparation.

FIG. 6 shows a schematic exemplar signal output for a real timesequencing operation using a two color/two amplitude signal set from anintegrated system of the invention where one trace (dashed) denotessignals associated with incorporation of A (high intensity signal) and T(lower intensity signal) bases, while the other signal trace (solidline), denotes the signals of a different emission spectrum, associatedwith G (high) and C (low) bases. The timing of incorporation and theidentity of the base incorporated, as derived from the color channel andintensity of the signal, are then used to interpret the base sequence.

Arrays of Integrated Optical Detection Devices

In order to obtain the volumes of sequence information that may bedesired for the widespread application of genetic sequencing, e.g., inresearch and diagnostics, higher throughput systems are desired. By wayof example, in order to enhance the sequencing throughput of the system,multiple complexes are typically monitored, where each complex issequencing a separate template sequence. In the case of genomicsequencing or sequencing of other large DNA components, these templateswill typically comprise overlapping fragments of the genomic DNA. Bysequencing each fragment, one can then assemble a contiguous sequencefrom the overlapping sequence data from the fragments.

As described above, and as shown in FIG. 1A, the template/DNApolymerase-primer complex of such a sequencing system is provided,typically immobilized, within an optically confined region, such as azero mode waveguide (ZMW), or proximal to the surface of a transparentsubstrate, optical waveguide, or the like. Preferably, such reactioncells are arrayed in large numbers upon a substrate in order to achievethe scale necessary for genomic or other large-scale DNA sequencingapproaches. Such arrays preferably comprise a complete integratedanalytical device, such as, for example, the devices shown in the blockdiagrams of FIGS. 2 and 5 . Examples of integrated systems comprisingarrays of optical analytical devices are provided in U.S. PatentApplication Publication Nos. 2012/0014837; 2012/0019828; and2012/0021525.

Arrays of integrated analytical devices, such as arrays of devicescomprising ZMWs, can be fabricated at ultra-high density, providinganywhere from 1000 ZMWs per cm², to 10,000,000 ZMWs per cm², or more.Thus, at any given time, it may be desirable to analyze the reactionsoccurring in 100, 1000, 3000, 5000, 10,000, 20,000, 50,000, 100,000, 1Million, 5 Million, 10 Million, or more ZMWs or other reaction regionswithin a single analytical system or even on a single substrate.

Using the foregoing systems, simultaneous targeted illumination ofthousands or tens of thousands of ZMWs in an array has been described.However, as the desire for multiplex increases, the density of ZMWs onan array, and the ability to provide targeted illumination of sucharrays, increases in difficulty, as issues of ZMW cross-talk (signalsfrom neighboring ZMWs contaminating each other as they exit the array),decreased signal:noise ratios arising from higher levels of denserillumination, and the like, increase. The devices and methods of theinstant invention address some of these issues.

The position on the detector upon which a given signal is incident isindicative of (1) the originating ZMW in the array, and (2) the emissioncharacteristics of the signal component, which is used, for example, toidentify the type of fluorescently labeled nucleotide analogincorporated in an extension reaction. As noted above, the detector mayinclude in some cases multiple sensing elements, each for detectinglight having a different color spectrum. For example, in the case ofsequencing, the sensor for each reaction cell may have 4 elements, onefor each of the four bases. In some cases, the sensor elements providecolor discrimination, in other cases, color filters are used to directthe appropriate color of light to the appropriate sensor element. Insome cases, the sensor elements detect intensity of signal only, withoutdiscriminating color. In some cases, the sensor elements identifies theincorporated nucleotide using a combination of emission characteristics.

Optical Waveguides

As mentioned above, the analytical devices of the instant invention, insome embodiments, comprise an optical waveguide to deliver excitationenergy to a sample. For example, as shown in FIG. 5 , optical waveguide506 conveys excitation light to the reaction region/well 502, where theevanescent field emanating from the waveguide 506 illuminates reactantswithin the reaction region 502. The use of an optical waveguide todeliver excitation illumination is advantageous for numerous reasons.For example, because the illumination light is applied in a spatiallyfocused manner, e.g., confined in at least one lateral and oneorthogonal dimension, using efficient optical systems, e.g., fiberoptics, waveguides, multilayer dielectric stacks (e.g., dielectricreflectors), etc., the approach provides an efficient use ofillumination (e.g., laser) power. For example, illumination of asubstrate comprising many separate reaction sites, “detection regions,”or “observation regions” using waveguide arrays as described herein canreduce the illumination power ˜10- to 1000-fold as compared toillumination of the same substrate using a free space illuminationscheme comprising, for example, separate illumination (e.g., via laserbeams) of each reaction site. In general, the higher the multiplex(i.e., the more surface regions to be illuminated on the substrate), thegreater the potential energy savings offered by the waveguideillumination schemes provided herein. In addition, since waveguideillumination need not pass through a free space optical train prior toreaching the surface region to be illuminated, the illumination powercan be further reduced.

In addition, because illumination is provided from within confinedregions of the substrate itself (e.g., optical waveguides), issues ofillumination of background or non-relevant regions, e.g., illuminationof non-relevant materials in solutions, autofluorescence of substrates,and/or other materials, reflection of illumination radiation, etc., aresubstantially reduced.

In addition to mitigating autofluorescence of substrate materials, thesystems described herein substantially mitigate autofluorescenceassociated with the optical train. In particular, in typicalfluorescence spectroscopy, excitation light is directed at a reaction ofinterest through at least a portion of the same optical train used tocollect signal fluorescence, e.g., the objective and other optical traincomponents. As such, autofluorescence of such components will contributeto the detected fluorescence level and can provide signal noise in theoverall detection. Because the systems provided herein direct excitationlight into the substrate through a different path, e.g., through anoptical fiber optically coupled to the waveguide in the substrate, thissource of autofluorescence is eliminated.

Waveguide-mediated illumination is also advantageous with respect toalignment of illumination light with surface regions to be illuminated.In particular, substrate-based analytical systems, and particularlythose that rely upon fluorescent or fluorogenic signals for themonitoring of reactions, typically employ illumination schemes wherebyeach analyte region must be illuminated by optical energy of anappropriate wavelength, e.g., excitation illumination. While bathing orflooding the substrate with excitation illumination serves to illuminatelarge numbers of discrete regions, such illumination may suffer from themyriad complications described above. To address those issues, targetedexcitation illumination may serve to selectively direct separate beamsof excitation illumination to individual reaction regions or groups ofreaction regions, e.g. using waveguide arrays. When a plurality, e.g.,hundreds or thousands, of analyte regions are disposed upon a substrate,alignment of a separate illumination beam with each analyte regionbecomes technically more challenging and the risk of misalignment of thebeams and analyte regions increases. Alignment of the illuminationsources and analyte regions may be built into the system, however, byintegration of the illumination pattern and reaction regions into thesame component of the system, e.g., a waveguide substrate. In somecases, optical waveguides may be fabricated into a substrate at definedregions of the substrate, and analyte regions are disposed upon thearea(s) of the substrate occupied by the waveguides.

Finally, substrates used in the waveguides may be provided from ruggedmaterials, e.g., silicon, glass, quartz or polymeric or inorganicmaterials that have demonstrated longevity in harsh environments, e.g.,extremes of cold, heat, chemical compositions, e.g., high salt, acidicor basic environments, vacuum and zero gravity. As such, they providerugged capabilities for a wide range of applications.

Waveguide substrates used in the devices and methods of the presentinvention generally include a matrix, e.g., a silica-based matrix, suchas silicon, glass, quartz or the like, polymeric matrix, ceramic matrix,or other solid organic or inorganic material conventionally employed inthe fabrication of waveguide substrates, and one or more waveguidesdisposed upon or within the matrix, where the waveguides are configuredto be optically coupled to an optical energy source, e.g., a laser. Suchwaveguides may be in various conformations, including but not limited toplanar waveguides and channel waveguides. Some preferred embodiments ofthe waveguides comprise an array of two or more waveguides, e.g.,discrete channel waveguides, and such waveguides are also referred toherein as waveguide arrays. Further, channel waveguides can havedifferent cross-sectional dimensions and shapes, e.g., rectangular,circular, oval, lobed, and the like; and in certain embodiments,different conformations of waveguides, e.g., channel and/or planar, canbe present in a single waveguide substrate.

In typical embodiments, a waveguide comprises an optical core and awaveguide cladding adjacent to the optical core, where the optical corehas a refractive index sufficiently higher than the refractive index ofthe waveguide cladding to promote containment and propagation of opticalenergy through the core. In general, the waveguide cladding refers to aportion of the substrate that is adjacent to and partially,substantially, or completely surrounds the optical core. The waveguidecladding layer can extend throughout the matrix, or the matrix maycomprise further “non-cladding” layers. A “substrate-enclosed” waveguideor region thereof is entirely surrounded by a non-cladding layer ofmatrix; a “surface-exposed” waveguide or region thereof has at least aportion of the waveguide cladding exposed on a surface of the substrate;and a “core-exposed” waveguide or region thereof has at least a portionof the core exposed on a surface of the substrate. Further, a waveguidearray can comprise discrete waveguides in various conformations,including but not limited to, parallel, perpendicular, convergent,divergent, entirely separate, branched, end-joined, serpentine, andcombinations thereof.

A surface or surface region of a waveguide substrate is generally aportion of the substrate in contact with the space surrounding thesubstrate, and such space may be fluid-filled, e.g., an analyticalreaction mixture containing various reaction components. In certainpreferred embodiments, substrate surfaces are provided in apertures thatdescend into the substrate, and optionally into the waveguide claddingand/or the optical core. In certain preferred embodiments, suchapertures are very small, e.g., having dimensions on the micrometer ornanometer scale, as described further below.

It is an object of devices and methods of the invention to illuminateanalytes (e.g., reaction components) of interest and to detect signalemitted from such analytes, e.g., by excitation and emission from afluorescent label on the analyte. Of particular interest is the abilityto monitor single analytical reactions in real time during the course ofthe reaction, e.g., a single enzyme or enzyme complex catalyzing areaction of interest. The waveguides described herein provideillumination via an evanescent field produced by the escape of opticalenergy from the optical core. The evanescent field is the optical energyfield that decays exponentially as a function of distance from thewaveguide surface when optical energy passes through the waveguide. Assuch, in order for an analyte of interest to be illuminated by thewaveguide, it must be disposed near enough to the optical core to beexposed to the evanescent field. In preferred embodiments, such analytesare immobilized, directly or indirectly, on a surface of the waveguidesubstrate. For example, immobilization can take place on asurface-exposed waveguide, or within an aperture in the substrate. Insome preferred aspects, analyte regions are disposed in apertures thatextend through the substrate to bring the analyte regions closer to theoptical core. Such apertures may extend through a waveguide claddingsurrounding the optical core, or may extend into the core of thewaveguide.

In certain embodiments, such apertures also extend through a mask layerabove the surface of the substrate. In preferred embodiments, suchapertures are “nanoholes,” which are nanometer-scale holes or wells thatprovide structural confinement of analytic materials of interest withina nanometer-scale diameter, e.g., ˜10-100 nm. In some embodiments, suchapertures comprise optical confinement characteristics, such aszero-mode waveguides, which are also nanometer-scale apertures and arefurther described elsewhere herein. Although primarily described hereinin terms of channel waveguides, such apertures could also be constructedon a planar waveguide substrate, e.g., where the planar waveguideportion/layer is buried within the substrate, i.e., is notsurface-exposed. Regions on the surface of a waveguide substrate thatare used for illumination of analytes are generally termed “analyteregions”, “reaction regions”, or “reaction sites”, and are preferablylocated on a surface of the substrate near enough to an optical core tobe illuminated by an evanescent wave emanating from the optical core,e.g., on a surface-exposed waveguide or at the bottom of an aperturethat extends into the substrate, e.g., into the waveguide cladding orcore. The three-dimensional area at a reaction site that is illuminatedby the evanescent field of a waveguide core (e.g., to an extent capableof allowing detection of an analyte of interest) is generally termed the“observation volume” or “illumination volume”. A region of a waveguidesubstrate that comprises one or more analyte regions is generallyreferred to as a “detection region” of the substrate, and a singlesubstrate may have one or multiple detection regions. Examples of suchoptical waveguides are provided in in U.S. Pat. No. 7,820,983 and U.S.Patent Application Publication No. 2012/0085894, as described above.

The present invention provides devices for waveguide-based illuminationof analyte regions in apertures (e.g., nanoholes or ZMWs) that in somecases reduce the variation in illumination, for example, by mitigatingpropagation losses over the length of the waveguide. Such propagationlosses can be further exacerbated by a metal layer disposed over thesurface of the substrate, because it can absorb optical energy from asurface-exposed or core-exposed waveguide, or even a waveguide near tothe surface of the substrate. Such metal layers are typically found inconventional ZMW arrays, presenting a challenge for combining sucharrays with waveguide illumination strategies.

One of the limitations of waveguide illumination is optical attenuationas the light propagates down the guide resulting in a reduction in powerat different locations in the guide. For example, a particular laserintensity coupled into the waveguide will experience a slow decrease inenergy density as light travels down the guide due to propagationlosses, with the highest power at the end nearest the illuminationsource and the lowest at the end farthest from the illumination source.The degree of the propagation loss is typically a function of thedesigned geometry and manufacturing tolerances, and presents a challengeto performing multiplexed analytical reactions because it constrains thespatial range of the usable waveguide structure. It is important tomaximize the distance over which the laser intensity is sufficientlyuniform, in order to maximize the multiplex capabilities of the system.It is therefore an object of the present invention to provide uniformpower over the length of a waveguide, e.g., to promote uniformillumination of all reaction sites to be illuminated by the waveguide.

In certain embodiments, a waveguide is tapered such that the coregradually becomes thinner along the direction of propagation. Thiscauses the degree of light confinement to be gradually increased, whichcan offset the gradual reduction in the total amount of power in theguide due to propagation losses and essentially maintain a desired modeshape and field strength for the optical energy propagated over thelength of the waveguide core. In principle, the sum of propagationlosses is balanced by the decreasing core size such that uniformity ofevanescent field strength can be held constant for an arbitrarily longdistance, with limitations to the strength of the evanescent field alsobeing dependent on the starting laser power and the starting waveguidecore dimensions. For a given waveguide substrate, once the propagationloss is determined the waveguide geometry can be designed to smoothlyvary, thereby modifying the degree of confinement such that the relativefield strength at the point of interest increases at the same rate thatpropagation losses reduce the total power in the guide. For example, atapered waveguide core can be widest at the portion most proximal to thelight source, slowly narrowing along the guide, with the fieldlocalization increasing at the same rate that propagation losses arereducing the waveguide field strength. The tapering can take place inany direction including the z direction (top to bottom), the y direction(side to side), or a combination thereof.

In certain embodiments, a waveguide cladding above a waveguide core in awaveguide substrate is tapered such that the waveguide core is slowlybrought closer to the reaction sites at the surface of the substrate byan ever-decreasing width of the waveguide cladding layer that separatesthe core from the reaction sites. As such, although there is propagationloss from the waveguide, as the field strength in the waveguidedecreases, it is brought closer to the reaction sites, and thisincreasing proximity compensates for an overall reduced field strength.In some embodiments, both the waveguide cladding layer and waveguidecore are tapered to mitigate loss of field strength due to propagationlosses.

Local Field Enhancement

In certain embodiments, the waveguides used in some of the devicesdisclosed herein comprise a specific feature associated with eachaperture, a local field enhancement element, to increase the efficiencyof sample illumination. Such a feature serves to improve the couplingefficiency between the waveguide and the illumination volume,particularly when the apertures are subwavelength apertures in a metallayer disposed over the surface of the substrate. For example, in someembodiments, the local field enhancement element is a layer of amaterial in the vicinity of each aperture that has a higher dielectricconstant than the cladding layer or that is a metal, such as, forexample, copper, silver, gold, or aluminum. One example of this type oflocal field enhancement element is shown in FIG. 7A, where the localfield enhancement element corresponds to a ring or other geometricpattern of high dielectric material or metal 706 surrounding theaperture 702, just below the opaque, metal layer 704. As shown in FIG.7A, a pattern of high dielectric material, such as Al₂O₃, or a metal,couples energy from the waveguide core of high dielectric 708, such asSi₃N₄, through the cladding of low dielectric 710, such as SiO₂. Othersuitable materials of high dielectric material may be substituted forAl₂O₃, as would be understood by those of ordinary skill in the art.

The material of high dielectric or metal surrounding the aperture mayserve other purposes in addition to improving the coupling of excitationlight to the illuminated volume within the aperture. For example, ifthis material is deposited such that it is exposed to the solution to beanalyzed, it may act as a distinct surface for immobilization ofbiomaterials or to prevent immobilization of those materials.Specifically, there may be advantages in providing different surfaces onthe bottom and sides of the aperture to allow, for example, for biasedimmobilization of reaction components. See, e.g., U.S. PatentPublication No. 2012/0085894. For example, as shown in FIG. 7A, thesides of the nanowell/aperture may expose a material such as, e.g., thehigh dielectric material or the metal, whereas the bottom of thenanowell/aperture may expose the cladding material, e.g., glass.Selective deposition of analytes is preferably effected when there arechemical differences between the surfaces, as would be understood bythose of ordinary skill in the art.

The local field enhancement element may include additional features tofurther enhance the efficiency of sample illumination by excitationlight, to further decrease propagation losses of excitation light, or toprovide other functions, such as, for example, enhancing the detectionof light emitted from the sample. One such additional feature isexemplified in FIG. 7B, where a pattern of high dielectric material ormetal 726 surrounds the nanowell/aperture 722 below the opaque, metallayer, 724. These patterns, for example a pattern of concentric ringssurrounding the aperture, as shown in FIG. 7B, may act as a broad areacoupler for the excitation light into the sample volume. They mayadditionally act to collimate light emitted from sample in theilluminated volume within the aperture, thus improving the detectabilityof that light. In this particular embodiment, the local enhancementelement thus serves to couple light both into and out of the aperture.Also shown in FIG. 7B is the waveguide core of high dielectric 728, suchas Si₃N₄, set within the cladding of low dielectric 730, such as SiO₂.

Aperture shapes, and the shape of the high dielectric material or metalsurrounding the apertures, may additionally enhance the coupling oflight energy from the waveguide core to the illuminated volume. See, forexample, Sahin et al. (2011) J. Nanophoton. 5(1), 051812;doi:10.1117/1.3599873. Subwavelength aperture shapes, including C-shapedapertures, triangle pairs, or diamond-shaped aperture structures mayaccordingly be usefully employed as local field enhancement elementsaccording to this aspect of the invention.

In yet another embodiment, the local field enhancement elementcorresponds to increasing the thickness of the cladding in the regionsof the waveguide that are not adjacent to the aperture and/or decreasingthe thickness of the cladding in the regions of the waveguide that areadjacent to the aperture. This embodiment provides, for example, fordecreased propagation losses, due to the increased distance between thelight beam and the metallic layer, and for increased couplingefficiency, due to the positioning of the aperture closer to theevanescent wave. As shown in FIG. 7C, decreasing the thickness of thecladding 750 (e.g., SiO₂) in the region around the aperture 742 byrecessing the aperture improves the coupling of light energy into theillumination volume. Increasing the thickness of the cladding in regionsremote from the aperture decreases propagation losses resulting frominteractions of the evanescent wave with the opaque metallic layer 744.FIG. 7C also shows an additional optional local field enhancementelement in the form of a ring of high dielectric material or metal 746surrounding the aperture below the opaque metallic layer. As notedabove, this element can be a ring or other pattern of high dielectricmaterial or metal in the vicinity of the aperture, just below the opaquemetallic layer. Combinations of different local field enhancementelements may thus provide synergistic improvements in the coupling oflight energy from the waveguide core to the illuminated volume and areconsidered within the scope of the invention. Also shown in FIG. 7C isthe waveguide core of high dielectric 748, such as Si₃N₄, set within thecladding of low dielectric 750.

FIGS. 8A and 8B illustrate top-down and cross-sectional views of anotherembodiment of the analytical device, similar to the device of FIG. 7C,in which the aperture is surrounded by a ring of high dielectric ormetal 806 and is recessed to bring it closer to the waveguide core.(Note that the opaque, metallic layer 804 is omitted from the top-downview shown in FIG. 8A.) This embodiment also includes an additionaloptional feature of this aspect of the invention, a stray-lighttermination element 812. Such a design feature can decrease backgroundsignal by blocking scattered light from the excitation source andpotentially also autofluorescence from materials within the device. Thematerial used in the stray-light termination element is selected frommaterials that selectively absorb light to be blocked from reaching thedetector layer of the device, as would be understood by one of ordinaryskill in the art. Also shown in FIGS. 8A and 8B is the waveguide core ofhigh dielectric 808, such as Si₃N₄, set within the cladding of lowdielectric 810, such as SiO₂. Exemplary dimensions for the device areindicated in FIG. 8A

FIGS. 9A-9C illustrate a device configuration used in a mathematicalsimulation of the effectiveness of the above designs, specificallyincreasing the cladding thickness in regions not adjacent to theaperture and including a ring of metal 906 in the vicinity of theaperture. (Note that the opaque, metallic layer 904 is omitted from thetop-down view shown in FIG. 9A.) In particular, the simulations involvea finite-difference time-domain (FDTD) solution of the Maxwellequations. See Taflove and Hagness (2004) Computational Electrodynamics:The Finite-Difference Time-Domain Method, Third Edition. FIGS. 9A-9Calso show the waveguide core of high dielectric 908, such as Si₃N₄, setwithin the cladding of low dielectric 910, such as SiO₂.

The simulations using the device configuration of FIGS. 9A-9Cdemonstrate that by increasing the cladding thickness between ZMWs by200 nm, the propagation loss, for example, due to proximity to themetallic layer, decreases from over 100 dB/mm to −14 dB/mm, allowing alower laser power to illuminate several ZMWs. Overall illuminationefficiency of the fluorophore is kept high, so as to keep the totallaser power in the waveguide low, thus helping to limit autofluorescencegenerated in the waveguide. In addition, a metal ring (or cylindricalshell) in the vicinity of the aperture provides enough metal to enhancethe coupling to the ZMW but not so much as to create waveguide loss.While any metal could be used, metals such as copper, silver, gold, andaluminum are preferred. Typical metal thicknesses (i.e., shellthickness) around the aperture are 100-400 nm. Since aperture diametersare preferably in the range of 100-200 nm, and most preferablyapproximately 140 nm, the outer diameter of the metal ring is thereforepreferably from 340 nm to 1 μm, although outer ring diameters of fromabout 0.2 to 2 μm, or even higher, are contemplated.

Dimensional Modulation of Waveguides

As noted above, efficient coupling of excitation light into theillumination volume requires that the optical waveguide be sufficientlyclose to the nanometer-scaled apertures, but placement of the opticalwaveguide too close to the metallic layer in which the apertures aredisposed may cause propagation losses. In order to overcome theselimitations, in another aspect of the invention, the optical waveguideis placed sufficiently far from the metallic layer to avoid propagationlosses, and the cross-sectional area of the waveguide is modulated inthe vicinity of the apertures to enlarge the mode size and thus increasethe coupling of light into the illuminated volume. In general, waveguidecore dimensions are designed to satisfy the single-mode condition, aswell as confining the mode compactly around the core region. Inpreferred embodiments, the cross-sectional area of the optical core isdecreased at locations where the optical waveguide illuminates theapertures. In some embodiments, the decrease in cross-sectional area ofthe optical core is achieved using adiabatic tapers in order to avoidextra power loss. In preferred embodiments, and as illustrated in FIGS.10A and 10B, the thickness of the optical waveguide is kept constant,while the cross-sectional area of the optical core is modulated byvarying the width of the optical core. Specifically, FIG. 10A provides atop-down view of waveguide 1008 with cross-sectional views of a normalsection of the waveguide with compact mode size (left cross-section) anda tapered-down section of the waveguide with expanded mode size (rightcross-section). FIG. 10B provides a three-dimensional perspective of theexemplary device showing locations of nanowell/apertures 1002 within theopaque, metal layer 1004 covering the array. Also shown aredimension-modulated waveguides 1008 positioned below thenanowell/apertures. Fabrication of the core layer in this embodiment issimplified, because the core thickness is uniform. In some embodiments,the optical signal passing through the waveguide core is transverseelectric (TE) polarized light. These embodiments provide advantages whenthe cross-sectional area of the waveguide is modulated by varying thewidth of the waveguide core, because the mode size of TE-polarized lightis most strongly affected by the waveguide width.

In variants of the just-described aspect of the invention, the width ofthe optical waveguide is kept constant, while the cross-sectional areaof the optical core, and thus the mode size of the transmitted light, ismodulated by varying the thickness of the optical core. As above, thecross-sectional area of the optical core is decreased at locations wherethe optical waveguide illuminates the apertures in order to maximizecoupling to the illuminated volume. In some embodiments, the opticalsignal passing through the waveguide core is transverse magnetic (TM)polarized light, since the mode size of TM-polarized light is moststrongly affected by the waveguide thickness, as would be understood byone of ordinary skill in the art.

In some embodiments of the invention, dimensional modulation of thewaveguides and local field enhancements may be usefully combined. Forexample, modulation of the cross-sectional area of the optical core ateach aperture may be usefully combined with a pattern of high dielectricmaterial or metal in the vicinity of each aperture, for example, belowthe metallic layer. Such combinations provide yet further improvement inthe efficiency of coupling of optical energy from the waveguide to theilluminated volume.

By way of non-limiting example, the typical waveguide widths for use inthe analytical devices of the instant invention range from 100 nm to1000 nm. Such widths therefore correspond to roughly 0.3 to 3.0wavelengths in a situation where the wavelength of excitation lightpropagated along the waveguide is 335 nm. (It should be noted that thewavelength of photons in a waveguide may be significantly shifted fromthat of the same photons traveling through air.) In some embodiments ofthe invention, the width of the waveguide optical core is decreased by 5to 90% at locations where the evanescent field illuminates thenanometer-scale apertures, compared to locations where the evanescentfield does not illuminate the apertures (in other words, the opticalcore is decreased from 0.15 wavelengths to 2.7 wavelengths for a corethat is 3.0 wavelengths wide). In some embodiments, the width of thewaveguide optical core is decreased by 5 to 50% at locations where theevanescent field illuminates the apertures, compared to locations wherethe evanescent field does not illuminate the apertures (i.e., from 0.15wavelengths to 1.5 wavelengths in a 3.0 wavelength wide core). Inpreferred embodiments, the width of the waveguide optical core isdecreased by 10 to 20% at those locations (i.e., from 0.30 wavelengthsto 0.60 wavelengths). As noted above, it is desirable in the devices ofthis aspect of the invention for the changes in width/cross-sectionalarea to be gradual, preferably adiabatic, in order to avoid anadditional mechanism for propagation losses. Such gradualtapering—narrower in the locations near the nanometer-scale aperturesand wider in the locations away from the nanometer-scale apertures—canbe readily be optimized in the design of the device, using analyticalcalculations, as would be understood by one of ordinary skill in theart.

Waveguide Core Positioning

In another aspect of the invention, the configuration and positioning ofthe waveguide core is varied in order to maximize collection of signalphotons while suppressing collection of background photons (e.g.,scattered light and autofluorescence). In particular, in theseembodiments the waveguide core is positioned so that it is not directlybetween the nanometer-scaled apertures and their correspondingdetectors. In preferred embodiments, the illuminated volume in eachaperture is illuminated by evanescent fields emanating from two or moreoptical cores. In some embodiments, the device further contains anopaque layer disposed between the waveguide layer and the detectorlayer. The opaque layer contains a plurality of openings positioned toallow signal photons from the sample to pass unimpeded into thedetector. The opaque layer would thus decrease access of photons from,for example, autofluorescence or scattering emanating from the waveguidecores, to the detector, and thus decrease background signal. As would beunderstood by one of ordinary skill in the art, signal photon collectionis defined by photon flux through the opening in the opaque material,and collection efficiency is modulated by the dimensions of the openingand the optical distance from the signal source. Likewise, collection ofautofluorescence and scattered light from the waveguide core through theopening is governed by the dimensions of the opening and the solid angleoccupied by the waveguide as viewed from the opening.

By using a waveguide that is not positioned directly between thenanometer-scaled aperture and the detector, i.e., a “slot” waveguide,the waveguide core, and associated autofluorescence and scattering, ismoved away from a direct path to the detector, thus increasing the angleof incidence into the detector and decreasing background signal.Furthermore, slot waveguides may provide increased optical fields in theregions of low refractive index between the high-index cores, and thusincrease coupling of optical energy from the waveguides to theilluminated volume. See, for example, Feng et al. (2006) IEEE J. QuantumElectron. 42, 885; Sun et al. (2007) Optics Express 15, 17967. Opticalsensing devices making use of slot waveguides have been described. See,for example, Barrios (2006) IEEE Photon Technol. Lett. 18, 2419; Barrioset al. (2007) Optics Letters 32, 3080; Barrios et al. (2008) OpticsLetters 33, 708; Robinson et al. (2008) Optics Express 16, 4296. Thesignal-to-background ratio in devices of the instant invention thatutilize a slot waveguide format may be optimized, for example, byvarying the dimension of the openings in the opaque layer and by varyingthe configuration of the waveguide (e.g., spacing between separateoptical cores and distance between waveguide core, opaque layer, anddetector layer), as would be understood by those of ordinary skill inthe art.

FIGS. 11A and 11B illustrate an example of this aspect of the inventionthat includes an optional local field enhancement element to increasefurther the coupling of excitation energy to the illuminated volume. Inparticular, FIG. 11A, illustrates a cross-sectional view of the devicedown the length of the divided (i.e., “slot”) waveguide cores 1108,surrounded by cladding of low dielectric 1110. As shown, the evanescentwaves emanating from the separate cores overlap and jointly illuminatethe sample “signal source” 1103 within a nanowell/aperture at the top ofthe drawing. The local field enhancement element 1106 is illustrated inthis drawing as a rectangle below the sample. This element could, forexample, correspond to a material of high dielectric constant or metalpatterned in the area adjacent to the nanometer-scale apertures, asdescribed above. As also described more fully above, this element servesto increase coupling between the waveguide core and the sample withinthe nanometer-scale apertures and thus increase signal emission from thesample. Such increased coupling could further increase thesignal-to-background ratio in the devices of the invention by decreasingthe size of the field necessary in the core and thus decreasing theassociated autofluoresence and scattering.

FIG. 11A further illustrates the opaque layer 1114, which is positionedbetween the waveguide layer and the detector 1118, and the opening inthe opaque layer to allow emission signal from the illuminated volume topass to the detector. The opaque layer is fabricated from any materialsuitable for attenuating transmission of excited light from thewaveguide core to the detector, e.g., a metal layer of sufficientthickness. As shown in FIG. 11A, the positioning of the waveguide coresaway from a direct alignment between the nanometer-scale aperture (notshown, but surrounding the illuminated sample volume) and the detectordoes not greatly diminish the efficiency of excitation of theilluminated volume, particularly if an optional local enhancementelement is included to enhance the coupling, but causes a significantdecrease in the transmission of autofluorescence or scattered light tothe detector, due to the presence of the opaque layer.

FIG. 11B provides a top-down view of the analytical device shown in FIG.11A, including the divided waveguide cores 1108. The “signal source”1103, which corresponds to the illuminated volume of the sample, isabove the plane of the drawing, and the opaque layer, including the“opening” 1116 in the opaque layer over the detector is below the planeof the drawing. The local enhancement element 1106 is illustrated as arectangular block, but any of the local enhancement elements describedabove could usefully be included in the device to enhance coupling ofexcitation energy to the sample volume. The detector is not shown inthis drawing but would be positioned below the opening and in line withthe signal source.

Waveguide Frequency Conversion

As noted above, excitation light is typically provided to sample volumesvia guided optics. Part of the motivation for this approach lies in thepossibility of dispensing with the disadvantages of classic free-spaceoptics by directing the sum total of excitation light needed to excitethe illumination volumes in all of the nanometer-scale apertures througha single optical system and onto a single chip. The guided opticalapproach can involve some disadvantages of its own, however, includingalignment complexity, cumulative autofluorescence, scattering, andcumulative laser heating. The latter three issues in particular arestraightforward limitations that result from the material properties ofthe optical system. The traditional chemistry and chemical formulationsused in fluorescence-based nucleic acid sequencing contribute to thedifficulties, as photonic excitation must be delivered within a fairlynarrow range of wavelengths, and the resulting emission wavelengths areonly slightly longer. Consequently, autofluorescence of the systemgenerally occurs within the same region of the spectrum as the desiredfluorescence emission signals and can therefore be a significantlimitation on the optical signal-to-noise ratio that can be achieved ina given system. Scattering can also be a detriment to signal-to-noise,because it is difficult to control the direction that scattered lighttravels as it leaves the guided mode, and some fraction may arrive at asensor, adding noise. Such scattered light may be difficult to filterout based on its wavelength. Similarly, photonic heating caused bynon-negligible absorption of the materials used to construct the opticalsystem and the sample chip is a strong function of the excitationwavelength, and there is little flexibility in altering this parametergiven the limitations in reagents used in typical sequencing reactions.

Accordingly, in another aspect of the invention, the limitations of atypical multiplexed sequencing system are addressed by using opticaltechniques such as harmonic generation, four-wave mixing, and stimulatedraman scattering to manipulate the wavelengths of pump excitation awayfrom those necessary for sample illumination and detection and towardspectral regions that are more suitable for the optical system,particularly with respect to the effects of the excitation photons onautofluorescence, laser heating, propagation loss, signal-to-backgroundratios, etc. Specifically, it is generally beneficial for excitationlight to be transported as longer-wavelength photons, for example asinfrared photons, which generate less autofluorescence within the deviceand which result in less laser heating. Shorter-wavelength light can begenerated at predetermined locations, as desired, preferably only in thelocations necessary to excite the relevant samples. Waveguide frequencyconversion can be effected by only slight modifications in opticalparameters through phase matching, or it can be actively switched on andoff through electro-optical effects that modify the refractive index ofone or more materials. Thus, light can be transported as an infraredpump and then be efficiently coupled into shorter wavelength harmonicwaveguide modes as desired. An additional advantage of such delivery ofwaveguide light is that scattering of light is dramatically reduced,because the infrared pump wavelength is significantly different inwavelength from the signal being collected by the detector, therebyreducing the detrimental impact of scattering on the signal-to-noiseratio.

Wavelength conversion in waveguides is typically effected through secondharmonic generation (SHG), wherein efficient conversion involves threefeatures: a nonlinear optical (NLO) material (in the case of SHG, forexample, a noncentrosymmetric material that responds to electromagneticfields with higher polarization multipoles), phase matching (typicallywith equal group velocities on both propagating modes), and sufficientoverlap integral (for example, where the energy density overlaps betweenthe fundamental mode, the harmonic mode, and the nonlinear material inthe structure). All three features can be designed into a waveguidestructure, and all three can be selected or adjusted by a variety oftechniques that are well understood by those of ordinary skill in theart. Furthermore, the techniques are widely available and are alreadybeing used in numerous commercial applications. In addition to SHG,similar techniques have been applied to other nonlinear opticalinteractions including optical parametric amplification (OPA) andstimulated Raman scattering. Such alternative approaches should also beconsidered within the scope of the instant invention.

At a fundamental level, periodic poling may be used to determine wherelight is converted from longer wavelengths, for example infraredwavelengths, to wavelengths usefully utilized in the direct excitationof samples, for example visible wavelengths. Such periodic poling may bedivided generally into two branches, a fixed periodic poling, whichwould not change in time, and a dynamic periodic poling, which can beused to fine-tune the wavelength conversion and to switch on or off theconversion at any location or set of locations, and at any time. Theprogrammability of such approaches is of particular value in theapplication of periodic poling to wavelength conversion for use in thesequencing methods described herein. Application of these techniques,including, for example, materials used, methods of fabrication, opticalproperties, theoretical principles, methods of tuning, conversionefficiencies, and so forth, are known in the art. See, for example, Yaoand Wang, Quasi-Phase-Matching Technology, in Nonlinear Optics andSolid-State Lasers, Springer Series in Optical Sciences 164,Springer-Verlag Berlin Heidelberg 2012. Specific examples of the use offixed and dynamic periodic poling in wavelength conversion devices havealso been reported. See Laurell et al. (2012) Optics Express 20, 22308;Chen et al. (2012) Optics Letters 37, 2814; Nava et al. (2010)Electronics Letters 46, 1686; Pan et al. (2010) Optics Communications284, 429.

An example of a basic SHG waveguide usefully employed in the devices ofthe instant invention is illustrated in FIG. 12A, where the nonlinearmedium is present in the core material of the waveguide, thussimplifying the overlap integral. An alternative structure, wherein thenonlinear medium is present in part of the cladding material, is shownin FIG. 12B.

FIG. 13A illustrates phase matching by periodic NLO materials. Such anapproach can allow significantly relaxed fabrication tolerances comparedto situations where phase matching is not provided by such periodicity.FIG. 13B illustrates phase modulation by electro-optic effects. Itshould be noted in this context that virtually all SHG materials alsoexhibit the strong Pockels coefficients that are important for thiselectro-optical effect.

The approaches for waveguide frequency conversion described herein canbe incorporated into the architecture of an analytical device in avariety of ways. For example, as shown in FIG. 14A, SHG conversion maytake place just after light is coupled into the chip. Alternatively, orin addition, excitation light may be injected into the waveguide andconverted into SHG at predetermined locations, as shown in FIG. 14B. Insome embodiments, excitation light can be programmed such that differentregions of nanometer-scale apertures and their corresponding illuminatedvolumes can be switched on and off independently.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein.

While specific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined by reference to theappended claims, along with their full scope of equivalents.

What is claimed is:
 1. An analytical device comprising: an opticalwaveguide comprising an optical core and a cladding; a metallic layerdisposed on a surface of the cladding; a plurality of nanometer-scaleapertures disposed in the metallic layer above the optical waveguide,such that optical energy passing through the optical core of the opticalwaveguide illuminates the plurality of apertures by an evanescent fieldemanating from the waveguide, wherein the plurality of apertures do notextend into the optical core of the optical waveguide; and a pluralityof local field enhancement elements associated with the plurality ofapertures, wherein the plurality of local field enhancement elements arepositioned between the optical core and the metallic layer.
 2. Theanalytical device of claim 1, wherein the local field enhancementelements comprise a high dielectric material or metal, wherein the highdielectric material has a higher dielectric constant than silicondioxide.
 3. The analytical device of claim 2, wherein the highdielectric material or metal is arranged in a geometric pattern aroundthe apertures.
 4. The analytical device of claim 3, wherein thegeometric pattern is selected from the group consisting of a circle, aseries of concentric circles, a C aperture, a triangle pair, and adiamond.
 5. The analytical device of claim 2, wherein the highdielectric material or metal is Al₂O₃, copper, silver, gold, oraluminum.
 6. The analytical device of claim 5, wherein the highdielectric material or metal is Al₂O₃.
 7. The analytical device of claim5, wherein the high dielectric material or metal is copper.
 8. Theanalytical device of claim 1, wherein the apertures are recessed intothe cladding.
 9. The analytical device of claim 1, wherein the claddingis between the optical core and the metallic layer and has a firstthickness that is decreased at locations where the evanescent fieldilluminates the apertures.
 10. The analytical device of claim 9, whereinthe cladding is between the optical core and the metallic layer and hasa second thickness of from 150 to 300 nm at locations where theevanescent field does not illuminate the apertures.
 11. The analyticaldevice of claim 10, wherein the second thickness is about 200 nm. 12.The analytical device of claim 1, wherein the local field enhancementelements comprise the shape of the nanometer-scale apertures.
 13. Theanalytical device of claim 12, wherein the shape of the nanometer-scaleapertures is selected from the group consisting of a C aperture, atriangle pair, and a diamond.
 14. The analytical device of claim 1,wherein the analytical device further comprises a plurality of analytesdisposed in analyte regions within the plurality of nanometer-scaleapertures.
 15. The analytical device of claim 14, wherein the pluralityof analytes comprise a plurality of biological samples.
 16. Theanalytical device of claim 15, wherein the plurality of biologicalsamples comprise a plurality of nucleic acids.
 17. The analytical deviceof claim 1, wherein the analytical device comprises at least 1,000, atleast 10,000, at least 100,000, at least 1,000,000, or at least10,000,000 nanometer-scale apertures.